Sperm Subpopulations Research Articles

Nuclear degraded sperm subpopulation is affected by poor chromatin compaction and nuclease activity

There is an interest in the nuclear degraded sperm subpopulation because, although it is present in a low percentage in all semen samples, patient groups such as varicocele and rearranged genome carriers show high levels of these degraded spermatozoa. This study is designed with two objectives in mind: first, incubations of H2 O2 and nuclease on DTT-treated and untreated samples to show the aetiology of this subpopulation and second, assessment of the correlation between the protamine ratio and nuclear degraded spermatozoa. A very high increase in the nuclear degraded subpopulation has been found with nuclease incubation, and it is even higher when it has been merged with nuclear decompaction using DTT. Alternatively, incubation with H2 O2 with and without DTT did not show such a significant increase in nuclear degraded spermatozoa. The protamine ratio correlated with this subpopulation, showing, in patients, that poor nuclear compaction would turn the sperm susceptible to degradation. Then, the assessment of nuclear degraded spermatozoa might not be only a measure of DNA degradation but also an indicator of chromatin compaction in the spermatozoa. Different patient groups would fit this model for sperm nuclear degradation, such as varicocele patients, who show a high percentage of immature spermatozoa and nuclear degraded spermatozoa, and reorganised genome carriers, where reorganisation might also cause poor chromatin compaction on the sperm nucleus.

Subpopulation pattern of eel spermatozoa is affected by post-activation time, hormonal treatment and the thermal regimen.

There has been a marked reduction in natural stocks of eels (genus Anguilla) over the past 60 years, and the culture of eels is still based on the capture of very large quantities of juveniles. It is necessary to close the life cycle in captivity in order to ease the pressure on wild populations. The aims of the present study were to evaluate sperm subpopulations (through cluster analysis of computer-aided sperm analysis data) in the European eel (Anguilla anguilla) and to assess the effects of motility acquisition time after activation (i.e. at 30, 60 and 90s), the thermal regimen (i.e. 10°C (T10) or 15°C (T15) and up to 20°C, or constant at 20°C (T20)) and hormonal treatments (i.e. human chorionic gonadotropin (hCG), recombinant (r) hCG or pregnant mare serum gonadotropin (PMSG)) on these subpopulations. In all cases, we obtained three subpopulations of spermatozoa: low velocity and linear (S1); high velocity with low linearity (S2); and high velocity and linear (S3; considered high quality). Total motility and S1 were affected by acquisition time; thus, 30s is recommended as the standard time for motility acquisition. When eels were kept at 20°C (T20), motility data fitted quadratic models, with the highest motility and proportion of S3 between Weeks 8 and 12 after the first injection. Lower temperatures (T10, T15) delayed spermiation and the obtaining of high-quality spermatozoa (S3), but did not seem to alter the spermiation process (similar subpopulation pattern). Conversely, the hormonal treatments altered both the dynamics of the subpopulation pattern and the onset of spermiation (with PMSG delaying it). Total motility and the yield of S3 with the widely used hCG treatment varied throughout the spermiation period. However, using rhCG allowed us to obtain high-quality and constant motility for most of the study (Weeks 7-20), and the S3 yield was also higher overall (61.8±1.3%; mean ± s.e.m.) and more stable over time than the other hormonal treatments (averaging 53.0±1.4%). Using T20 and rhCG would be more economical and practical, allowing us to obtain a higher number of S3 spermatozoa over an extended time.

Low amounts of mitochondrial reactive oxygen species define human sperm quality

We have applied the mitochondria-specific superoxide fluorescent probe MitoSOX Red (MitoSOX) to detect mitochondria-specific reactive oxygen species (mROS) production in human sperm samples using flow cytometry. We show that human ejaculates are heterogeneous in terms of mROS production, with three subpopulations clearly detectable, comprising sperm that produce increasing amounts of mROS (MitoSOX-, MitoSOX+, and MitoSOX++). The sperm subpopulation producing the lowest amount of mROS represented the most functional subset of male gametes within the ejaculate, as it was correlated with the highest amount of live and non-apoptotic sperm and increased both in samples with better semen parameters and in samples processed by both density-gradient centrifugation and swim-up, both known to select for higher quality sperm. Importantly, the MitoSOX- subpopulation was clearly more prevalent in samples that gave rise to pregnancies following assisted reproduction. Our work, therefore, not only describe discreet human sperm heterogeneity at the mROS level but also suggests that mROS may represent a strategy to both evaluate sperm samples and isolate the most functional gametes for assisted reproduction.

Evaluation of sperm subpopulation structure in relation to in vitro sperm–oocyte interaction of frozen-thawed semen from Holstein bulls

The present study examined the relationship between the relative amount of high motile sperm and sperm–oocyte interactions obtained from Holstein bull ejaculates. Post-thaw sperm motility was analyzed using a computer-assisted sperm analyzer system and evaluated to determine the sperm motility subpopulations. Adhesion and penetration of zona pellucida (ZP) and pronucleus formation using post-thawed samples (15 ejaculates form 5 different bulls) with different percentages of sperm in the subpopulation with the fastest and most progressive subpopulation (subpopulation 4 [SP4]) were analyzed. The correlation between the proportion of sperm in SP4 and the number of spermatozoa bound to the zona pellucida (ZBA), the penetration rate, and the rate of pronucleus formation were calculated. A significant (P < 0.05) and positive correlation was found between the number of spermatozoa bound to the zona pellucida, the penetration rate, and the rate of pronucleus formation with the proportion of sperm in SP4 (r = 0.79, r = 0.66, and r = 0.63, respectively). Our results suggest that this specific high motile and progressive subpopulation is positively and significantly correlated with the ability of a thawed bull semen sample to interact properly with the oocyte and its extracellular vestments. These findings emphasize the relevance of analyzing semen subpopulation composition to predict bull sperm fertilizing ability and to select Holstein bulls for breeding purposes.

Colloid Centrifugation Selects Normal Spermatozoa from Polymorphic Bull Ejaculates: A Case Study

Semen from a Western Finncattle bull exhibiting a highly polymorphic spermiogram was processed by colloid centrifugation using Androcoll-B, a species-specific silane-coated silica colloid. In the first experiment, Single Layer Centrifugation (SLC) was used to identify which density colloids were needed to separate different cell populations. Colloids of the two chosen densities were then used in a density gradient resulting in two sperm subpopulations, one containing nearly all normally sized spermatozoa and the other enriched for the macrocephalic spermatozoa. Microcephalic spermatozoa did not appear in either of the selected subpopulations. Using a combination of SLC and DGC with this species-specific colloid, it was possible to separate the spermatozoa into different subpopulations, that is, a subpopulation containing nearly all normally sized spermatozoa, and another one enriched for the macrocephalic spermatozoa. Thus, colloid centrifugation could be used to select sufficient normal spermatozoa from a highly polymorphic ejaculate for AI, if desired.

Rapid selection of sperm with high DNA integrity

Sperm selection is essential to assisted reproductive technology (ART), influencing treatment outcomes and the health of offspring. The fundamental challenge of sperm selection is dictated by biology: a heterogeneous population of ~10(8) sperm per milliliter with a short lifetime in vitro. However, conventional sperm selection approaches result in less than 50% improvement in DNA integrity. Here, a clinically applicable microfluidic device is presented that selects sperm based on the progressive motility in 500 parallel microchannels. The result is a one-step procedure for semen purification and high DNA integrity sperm selection from 1 mL of raw semen in under 20 minutes. Experiments with bull sperm indicate more than 89% improvement in selected sperm vitality. Clinical tests with human sperm show more than 80% improvement in human DNA integrity, significantly outperforming the best current practices. These results demonstrate the presence of a sub-population of sperm with nearly intact chromatin and DNA integrity, and a simple clinically-applicable lab-on-a-chip method to select this population.

Sperm Population Structure and Male Fertility: An Intraspecific Study of Sperm Design and Velocity in Red Deer1

Sperm design and velocity play key roles in influencing sperm performance and, therefore, can determine fertilization success. Several interspecific studies have demonstrated how these features correlate, and it has been hypothesized that selection may drive changes in these sperm traits. Here, we examine the association between sperm design and swimming velocity in a study conducted at an intraspecific level in Iberian red deer (Cervus elaphus hispanicus). We addressed how the structure of different sperm subpopulations, based on sperm morphometry and velocity, are interrelated and, in turn, how they associate with fertility. Our results show that males with high fertility rates have ejaculates with high percentages of spermatozoa exhibiting fast and linear movements and that these are highly correlated with a large proportion of spermatozoa having small and elongated heads. On the other hand, males with low fertility are characterized by a subpopulation structure in which slow and nonlinear as well as small and wide spermatozoa are predominant. These findings provide insight regarding how sperm size and velocity are interrelated and how they both are associated with fertility.

Relationship between conventional semen characteristics, sperm motility patterns and fertility of Andalusian donkeys (Equus asinus)

Sperm quality has an important role in determining fertility. The aims of this study were to compare the conventional sperm parameters, plus the characteristics of the motility patterns of the different sperm subpopulations, of donkey donors with different fertility level, and to determine their relationships to fertility. Thirty ejaculates from 6 Andalusian donkeys were assessed for gel-free volume, pH, sperm concentration, motility and morphology. The fertility of donkeys was classified on the basis of pregnancy rates per cycle, where donkeys with a per cycle pregnancy rate ≥60% were considered to be “fertile” (n=3) and those with a per cycle pregnancy rate <40% were categorized to be “sub-fertile” (n=3). Significant differences (P<0.001) between the “fertile” and the “sub-fertile” group were found for total and progressive motility, and for straight line velocity. Sperm variables associated (P<0.05) with an increase in percent pregnant per cycle included total motility (r=0.37), progressive motility (r=0.53), curvilinear velocity (r=0.44), straightness (r=0.39), beat cross frequency (r=0.44), and gel-free volume (r=0.53). Four sperm subpopulations (sP) were identified in fresh semen: sP1 (slow and non-progressive spermatozoa, 20%), sP2 (moderately slow but progressive spermatozoa, 71.2%), sP3 (highly active but non-progressive spermatozoa, 2.9%), and sP4 (highly active and progressive spermatozoa, 5.9%). The lowest percentage (3.1%; P<0.001) of sP4 spermatozoa was observed in the “sub-fertile” group. Three of the sperm subpopulations were related (P<0.05) to fertility (sP2, r=0.54; sP3, r=0.45; sP4, r=0.56). In conclusion, we were able to relate the fertility of donkeys with in vitro measures of sperm motility using computer-assisted sperm analysis techniques.

Brown bear sperm double freezing: Effect of elapsed time and use of PureSperm® gradient between freeze–thaw cycles

The use of sexed spermatozoa has great potential to captive population management in endangered wildlife. The problem is that the sex-sorting facility is a long distance from the semen collection place and to overcome this difficulty two freeze–thaw cycles may be necessary. In this study, effects of refreezing on brown bear electroejaculated spermatozoa were analyzed. We carried out two experiments: (1) to assess the effects of the two freezing–thawing cycles on sperm quality and to analyze three different elapsed times between freezing–thawing cycles (30, 90 and 180min), and (2) to analyze the use of PureSperm between freezing–thawing cycles to select a more motile and viable sperm subpopulation which better survived first freezing. The motility, viability and undamaged acrosomes were significantly reduced after the second thawing respect to first thawing into each elapsed time group, but the elapsed times did not significantly affect the viability and acrosome status although motility was damaged. Our results with the PureSperm gradient showed higher values of viability in freezability of select sample (pellet) respect to the rest of the groups and it also showed a significant decrease in the number of acrosome damaged. In summary, the double freezing of bear semen selected by gradient centrifugation is qualitatively efficient, and thus could be useful to carry out a sex-sorting of frozen–thawed bear spermatozoa before to send the cryopreserved sample to a biobank. Given the low recovery of spermatozoa after applying a selection gradient, further studies will be needed to increase the recovery rate without damaging of the cell quality.

In vitro reconstruction of inflammatory reaction in human semen: effect on sperm DNA fragmentation

The study was aimed at evaluating an in vitro induction of DNA damage in three sperm subpopulations exposed to selected inflammatory mediators, such as leukocytes, two combinations of pro-inflammatory cytokines (interleukin [IL]-6+IL-8 and IL-12+IL-18) and two bacterial strains (Escherichia coli and Bacteroides ureolyticus). Semen samples from normozoospermic volunteers were differentiated by swim-up (swim-up fraction) and Percoll gradient procedures (90% and 47% Percoll fractions). Leukocytes were isolated from the whole heparinized blood using the density gradient centrifugation technique. DNA fragmentation in sperm fractions was evaluated using flow cytometry with TUNEL labeling and Comet assay. Out of the inflammatory factors tested, bacteria were found to have a greatest toxic effect on sperm DNA, especially in fractions isolated by Percoll gradient, compared with untreated cells (P<0.05). The results indicate that inflammatory mediators can be a direct cause of DNA fragmentation in ejaculated spermatozoa, which can ultimately lead to limited fertilizing abilities of the germ cells. In contrast to the swim-up technique, the selection of spermatozoa by gradient procedures increases the vulnerability of mature spermatozoa to the harmful effects of infectious agents on DNA integrity. This observation may have some meaning for recommendations concerning laboratory techniques used in assisted reproductive therapy.

Sperm guidance to the egg finds calcium at the helm

Sperm respond to multiple cues during guidance to the egg including chemical attractants, temperature, and fluid flow. Of these, sperm chemotaxis has been studied most extensively-over 100years-but only recently has it started to be understood at the molecular level. The long gestation in this understanding has largely been due to technical limitations that include the detection of calcium signal dynamics in a relatively small structure-the flagellum, measurement of actual chemoattractant gradients, the fact that only subpopulations of sperm respond at any given time, and the diversity in swimming behaviors that sperm exhibit from different species. Today, measurements of flagellar calcium signals on a fast time scale, discovery of the ion channels and organelles that may regulate these signals, and better understanding and quantitation of sperm swimming behaviors involved have given more certainty to our understanding of sperm directional swimming and its control by characteristic, calcium-directed asymmetric flagellar bends. Future research will need to apply these technical advances to other forms of sperm guidance such as thermotaxis and rheotaxis as well as gaining an understanding of how the flagellar apparatus is controlled by calcium.

Role of Sperm Velocity Variables Associated with Poultry Breed in ‘Last Male Precedence’

It is well known that when a hen mates with multiple roosters, it is the sperm of the last male that usually fertilizes most of the eggs ('last male precedence'). Sperm quality varies between males within a breed, but also between breeds, and thus, sperm competitiveness after mating may depend on the breeds of the roosters involved. The aim of the present work was to identify differences in sperm competitiveness between breeds, especially with respect to motility. A multibreed mating model was used. Blue Andaluza (BA) and Black Castellana (BC) hens left for 21days with BA and BC roosters, respectively, were then left with Black-barred Andaluza (Bb) roosters for another 21days (experimental groups hBA-rBC-rBb and hBC-rBA-rBb). Bb roosters (as the second breed replacing the first) fertilized the majority of eggs in both the hBC-rBA-rBb and hBA-rBC-rBb groups. The percentage of offspring sired by BA roosters (8.0%) was higher (p<0.05) than the percentage of chicks sired by BC roosters (2.1%). The fertility of the BC hens in the hBC-rBA-rBb group was higher (p<0.01) than that of the BA hens in the hBA-rBC-rBb group. No difference in sperm concentration was seen between the breeds. Within the rapid sperm subpopulation (sperm velocity, >50μm/s), Bb sperm showed a higher straight-line velocity (VSL) and average path velocity (VAP) (p<0.05) than BC sperm. The VSL and VAP values for Bb and BA sperm were similar. In conclusion, the present results show that the sperm of the BA breed, traditionally regarded as of moderate fertility, compensates for this drawback via sperm movement characteristics that afford it an advantage in competition scenarios involving males of other breeds. The VSL and VAP of the rapid sperm subpopulation may play the most important role in securing last male precedence.

Differential distribution of sperm subpopulations and incidence of pleiomorphisms in ejaculates of captive howling monkeys (Alouatta caraya)

The aim of this study was to develop an objective method to determine the incidence of pleiomorphisms and its influence on the distribution of sperm morphometric subpopulations in ejaculates of howling monkeys (Alouatta caraya) by using a combination of computerized analysis system (ASMA) and principal component analysis (PCA) methods. Ejaculates were collected by electroejaculation methods on a regular basis from five individuals maintained under identical captive environmental, nutritional, and management conditions. Each sperm head was measured for dimensional parameters (Area [A, (square micrometers)], Perimeter [P, (micrometers)], Length [L, (micrometers)], and Width [W, (micrometers)]) and shape-derived parameters (Ellipticity [(L/W)], Elongation [(L - W)/(L + W)], and Rugosity [(4лA/P (2))]). PCA revealed two principal components explaining more than the 96 % of the variance. Clustering methods and discriminant analyzes were performed and seven separate subpopulations were identified. There were differences (P < 0.001) in the distribution of the seven subpopulations as well as in the incidence of abnormal pleiomorphisms (58.6 %, 49.8 %, 35.1 %, 66.4 %, and 55.1 %, P < 0.05) among the five donors tested. Our results indicated that differences among individuals related to the incidence of pleiomorphisms, and sperm subpopulational structure was not related to the captivity conditions or the sperm collection method, since all individuals were studied under identical conditions. In conclusion, the combination of ASMA and PCA is a useful clinical diagnostic resource for detecting deficiencies in sperm morphology and sperm subpopulations in A. caraya ejaculates that could be used in ex situ conservation programs of threatened species in Alouatta genus or even other endangered neotropical primate species.

Fluorescence Technologies for Evaluating Male Gamete (Dys)Function

For many years, andrologists have sought ways of assessing sperm fertility, especially of new sires entering the breeding chain. As knowledge of the complex processes that enable sperm to fertilize eggs has increased, it has become clearer that quantitative estimation of the fertilizing potential of a sire or an ejaculate is actually unlikely ever to be fully realized. Here, we propose that a better approach is to identify substandard males and semen samples. During the past decades, the use of fluorescence technologies in biomedical science has burgeoned, with the development of very powerful instrumentation such as confocal microscopy and flow cytometers of ever-increasing capabilities together with a vast range of fluorochromes and fluorochrome conjugates. This technology has been applied to andrology but thus far in only a relatively simple way. In this review, we offer strategies for assessing a large range of sperm functions thought to be related to fertilizing ability over a temporal window rather than at a single time point. From such an assessment profile, sperm samples that over-respond or do not respond sufficiently could be identified, termed dysfunctional and rejected. We outline the rationales behind such tests, present information on new potentially useful fluorochromes and current flow cytometer models that would be suitable for the multicolour multifunctional tests we propose, and we offer suggestions as to how andrologists might design such multicolour tests for themselves.

Aneuploidy rates for chromosomes X/Y and 18 among preselected spermatozoa in men with severe teratospermia

Eight infertile men with various degrees of oligoasthenoteratozoospermia and repeated implantation failure were selected for this study due to exceptionally high rates of sperm aneupoidy in their ejaculates. All subjects had normal physical examination, karyotype and serum FSH concentration. Prior to IVF treatment, spermatozoa was collected, processed, micromanipulated and tested for chromosomes X, Y and 18 using fluorescence in-situ hybridization. Aneupoidy rates for chromosomes X, Y and 18 were determined among sperm population selected for normal morphology using high-order magnification light microscopy. A second group of fast motile spermatozoa were collected using an intracytoplasmic sperm injection pipette from the medium–oil interface from microdroplets. The average aneuploidy rates for the three chromosomes were 7.6% (395/5182) in the sperm specimen before selection, 8.7% (116/1326) in the normal morphology selected group and 4.3% (59/1388; P<0.001) in the fast motile selected group. In conclusion, high-magnification light microscopy aimed at selection of spermatozoa with normal morphology did not affect the aneuploidy rate. On the other hand, fast motile spermatozoa harboured significantly less chromosomal abnormalities (P<0.001). Preselection of the most rapid sperm subpopulation for intracytoplasmic sperm injection may improve the qualities of the fertilizing spermatozoon.Eight infertile men with various degrees of oligoasthenoteratozoospermia and repeated implantation failure were selected for the study due to exceptionally high rates of sperm aneupoidy in their ejaculates. All subjects had normal physical examination, karyotype and serum FSH concentration. Prior to IVF treatment, spermatozoa was collected, processed, micromanipulated and tested for chromosomes X, Y and 18 using fluorescence in-situ hybridization. Aneupoidy rates for chromosomes X, Y and 18 were determined among sperm population selected for normal morphology using high-order magnification light microscopy. A second group of fast motile spermatozoa were collected using an ICSI pipette from the medium–oil interface from microdroplets. The average aneuploidy rates for the three chromosomes were 7.6% (395/5182) in the sperm specimen before selection, 8.7% (116/1326) in the normal morphology selected group and 4.3% (59/1388, P<0.001) in the fast motile selected group. In conclusion, high-magnification light microscopy aimed at selection of spermatozoa with normal morphology did not affect the aneuploidy rate. On the other hand, the fast motile spermatozoa harboured significantly less chromosomal abnormalities. Pre-selection of the most rapid sperm subpopulation for ICSI may improve the qualities of the fertilizing spermatozoon.

Density gradient centrifugation prior to cryopreservation and hypotaurine supplementation improve post-thaw quality of sperm from infertile men with oligoasthenoteratozoospermia

Can selection of spermatozoa by density gradient centrifugation prior to cryopreservation and/or hypotaurine supplementation improve the post-thaw quality of sperm from infertile men with oligoasthenoteratozoospermia? Sperm selection by density gradient centrifugation before freezing and supplementation of the media by hypotaurine is beneficial for the cryopreservation of semen samples of patients with oligoasthenoteratozoospermia. Sperm from men with oligoasthenoteratozoospermia are more susceptible than normal to cryoinjury. Density gradient centrifugation before sperm freezing may allow the selection of a subpopulation of spermatozoa more resistant to cryopreservation. Hypotaurine is an antioxidant with a protective effect on sperm functions. The experiment was carried out according to a factorial design involving two binary factors resulting in four treatment combinations which were randomly allocated in oligoasthenoteratozoospermia sperm samples from 64 patients recruited between January 2009 and June 2010. Semen was provided by 64 men undergoing evaluation for infertility at the Centre for Reproductive Medicine of the University Hospital in Clermont-Ferrand, France, between January 2009 and June 2010. Four treatment combinations were tested: sperm freezing before selection without (F-S/H-; n = 16) and with hypotaurine supplementation (F-S/H+; n = 16); sperm selection before freezing without (S-F/H-; n = 16) and with hypotaurine supplementation (S-F/H+; n = 16). Measurements of sperm recovery rates and markers of apoptosis (externalization of phosphatidylserine (PS), mitochondrial membrane potentialand DNA fragmentation) were compared in recovered spermatozoa after each procedure. Higher recovery rates of progressive and total motile spermatozoa were observed when sperm selection was performed before freezing (P < 0.05). The protective effect of hypotaurine was only observed on the percentage of live spermatozoa with PS externalization among total live spermatozoa (AN+ PI-/((AN+ PI-) + (AN- PI-)) when the sperm selection by density gradient centrifugation was performed before freezing (S-F/H+ versus S-F/H-: 6.8 ± 1.09 versus 11.8 ± 2.03%, P = 0.04). The percentage of mitochondrial membrane potential (DiOC6(3) (high)) spermatozoa was higher (P = 0.001) when sperm selection was done before freezing compared with procedures in which sperm selection was done after sperm freezing with (S-F/H+ versus F-S/H+: 58.1 ± 3.50 versus 46.7 ± 5.48%) or without (S-F/H- versus F-S/H-: 57.0 ± 5.18 versus 35.4 ± 4.99%) hypotaurine supplementation. The percentages of TUNEL+ spermatozoa were significantly lower (P = 0.001) when sperm selection was done before sperm freezing compared with procedures in which sperm selection was done after sperm freezing with (S-F/H+ versus F-S/H+: 38.6 ± 9.59 versus 55.7 ± 5.88%) or without hypotaurine supplementation (S-F/H- versus F-S/H-: 37.2 ± 7.91 versus 71.0 ± 5.66%). The ICSI outcomes were not assessed and the fertility of the spermatozoa remains unknown. Sperm selection by density gradient centrifugation before freezing and hypotaurine supplementation could improve the cryopreservation of sperm from oligoasthenoteratozoospermic men and make a larger number of functional spermatozoa available for ICSI. This work was supported by a hospital grant (Projet Hospitalier Recherche Clinique, CHU Clermont Ferrand, France). None of the authors has any conflict of interest to declare.

Cluster analysis reveals a binary effect of storage on boar sperm motility function.

Storage of liquid-preserved boar spermatozoa is associated with a loss of fertilising ability of the preserved spermatozoa, which standard semen parameters barely reflect. Monitoring responses to molecular effectors of sperm function (e.g. bicarbonate) has proven to be a more sensitive approach to investigating storage effects. Bicarbonate not only initiates capacitation in spermatozoa, but also induces motility activation. This occurs at ejaculation, but also happens throughout passage through the oviduct. In the present study we tested whether the specific response of boar sperm subpopulations to bicarbonate, as assessed by motility activation, is altered with the duration of storage in vitro. Three ejaculates from each of seven boars were diluted in Beltsville thawing solution and stored at 17°C. Only minor changes in the parameters of diluted semen were revealed over a period of 72h storage. For assessment of bicarbonate responses, subsamples of diluted spermatozoa were centrifuged through a discontinuous Percoll gradient after 12, 24 and 72h storage. Subsequently, spermatozoa were incubated in two Ca2+-free variants of Tyrode's medium either without (TyrControl) or with (TyrBic) 15mM bicarbonate, and computer-aided sperm analysis motility measurements were made. Cluster analysis of imaging data from motile spermatozoa revealed the presence of five major sperm subpopulations with distinct motility characteristics, differing between TyrBic and TyrControl at any given time (P<0.001). Although there was an increasing loss of motility function in both media, bicarbonate induced an increase in a 'fast linear' cohort of spermatozoa in TyrBic regardless of storage (66.4% at 12h and 63.9% at 72h). These results imply a binary pattern in response of sperm motility function descriptors to storage: although the quantitative descriptor (percentage of motile spermatozoa) declines in washed semen samples, the qualitative descriptor (percentage of spermatozoa stimulated into fast linear motion by bicarbonate) is sustained independent of the duration of storage.

Picomolar gradients of progesterone select functional human sperm even in subfertile samples

More than 1 million infertility treatments are practiced around the world per year, but only 30% of the couples succeed in taking a baby home. Reproductive technology depends in part on sperm quality, which influences not only fertilization but also embryo development and implantation. In order to provide a better quality sperm subpopulation, innovative sperm selection techniques based on physiological sperm features are needed. Spermatozoa at an optimum state may be selected by following an increasing concentration gradient of picomolar progesterone, a steroid secreted by the cumulus cells at the time of ovulation. In this study we developed a method to recruit spermatozoa at the best functional state, based on sperm guidance toward progesterone. The sperm selection assay (SSA) consists of a device with two wells connected by a tube. One well was filled with the sperm suspension and the other with picomolar progesterone, which diffused inside the connecting tube as a gradient. The sperm quality after the SSA was analyzed in normal and subfertile semen samples. Several sperm parameters indicative of sperm physiological state were determined before and after the SSA: capacitation, DNA integrity and oxidative stress. After the SSA, the mean level of capacitated spermatozoa increased three times in normal and in subfertile samples. The level of sperm with intact DNA was significantly increased, while sperm oxidative stress was decreased after sperm selection. Interestingly, the exposure to a progesterone gradient stimulated the completion of capacitation in some spermatozoa that could not do it by themselves. Thus, the SSA supplies a sperm population enriched with spermatozoa at an optimum physiological state that may improve the assisted reproductive technology outcome.

Dynamics of sperm subpopulations based on motility and plasma membrane status in thawed ram spermatozoa incubated under conditions that support in vitro capacitation and fertilisation

The present study evaluated modifications occurring in thawed ram spermatozoa during incubation in different media that supported in vitro capacitation and fertilisation, and examines how these changes relate to IVF. Thawed sperm samples were incubated under capacitating (Cap) and non-capacitating (non-Cap) conditions for 0, 1 and 2h and used in an IVF test. During incubation, changes related to membrane status and the motility pattern of spermatozoa were assessed, the latter being used to characterise sperm subpopulations. A significantly greater increase (P≤0.05) in the percentage of spermatozoa with higher membrane fluidity was observed in samples incubated with Cap medium from the beginning of incubation. In addition, changes over time in the distribution of the motile subpopulation were particularly evident when spermatozoa were incubated with Cap medium, with a noted increase in spermatozoa classified as 'hyperactivated like', with major changes occurring after 1h incubation. Both characteristics (i.e. membrane fluidity and the percentage of the hyperactivated-like subpopulation) were significantly related with in vitro fertility, and only sperm samples incubated with the Cap medium were capable of fertilising oocytes. These results support the idea that changes in sperm membrane fluidity and motility pattern (i.e. an increase in hyperactivated spermatozoa) are needed for fertilisation to take place.

Characterisation of a subpopulation of sperm with massive nuclear damage, as recognised with the sperm chromatin dispersion test

Assessment of human sperm DNA fragmentation by the sperm chromatin dispersion (SCD) test is based on the detection of haloes of spreading DNA loops after sequential DNA denaturing and protamine removal. After the SCD test, sperm without DNA fragmentation show chromatin haloes emerging from the central nuclear core, while sperm containing fragmented DNA present small or no haloes. The nuclear degraded sperm are recognised as a differentiated category within the sperm with fragmented DNA, whose cores appear irregularly and/or faintly stained. This subpopulation is more prevalent in patients with varicocele. Protein staining with 2.7-dibrom-4-hydroxy-mercury-fluorescein demonstrated that degraded sperm intensely lose nuclear core proteins after the SCD processing. Moreover, degraded sperm are 65% more faintly labelled for DNA breaks after in situ nick translation (ISNT) on average, due to extensive DNA loss. A two-dimensional comet assay under sequential neutral and alkaline conditions demonstrated that degraded sperm contain both massive double- and single-strand DNA breaks. The degraded sperm appear as a subpopulation with stronger nuclear damage, affecting both DNA and protein fractions, possibly due to intense intratesticular oxidative stress, what could explain its higher proportion in patients with varicocele.